The type 1 angiotensin II receptor (AT1R) mediates the known physiological actions of the octapeptide hormone, angiotensin II (Ang II) on blood pressure regulation, aldosterone secretion, and sodium balance. Recently, Ang II activation of AT1 receptors in the cardiovascular system has been implicated in the development of atheroma and cardiac failure, in addition to its known involvement in arterial hypertension. Most of the actions of Ang II are mediated by coupling of the AT1R to Gq/11 proteins, followed rapidly by the initiation of phosphoinositide-calcium signaling and activation of PKC isoforms. This is associated with the onset of phosphorylation cascades to the nucleus, and increased expression of genes regulating cell growth, differentiation and function. Many of the growth-related actions of Ang II are mediated by transactivation of the EGF receptor (EGFR), which initiates ras-dependent stimulation of MAP kinase cascades. We have analyzed this process in C9 hepatic cells and AT1R-transfected COS-7 cells, in which Ang II-induced ERK phosphorylation proved to be largely dependent on transactivation of the EGFR and is independent of the endocytosis of the AT1R and EGFR. In C9 cells, Ang II-induced ERK activation is initiated by a PKCdelta-dependent but Ca2+-independent mechanism, and is predominantly mediated by the Src/Pyk2 complex through transactivation of the EGFR. Further investigations are addressing the nature of the interactions between GPCRs and receptor tyrosine kinases, and the extent to which caveolae and other cell membrane structures are involved in the signaling cross-talk between different types of plasma-membrane receptors. In a mutational analysis of the function of a highly conserved domain (DRYXXV/IXXPL) in the second intracellular loop of the AT1R, individual Ala replacements of the Asp125 and Arg126, but not of Tyr127, moderately impaired agonist-induced inositol phosphate signaling. However, concomitant Asp/Arg substitutions markedly reduced both inositol phosphate signaling and AT1R internalization. Alanine-scanning showed that Ile130, His132, and Pro133 substitutions reduced agonist-induced inositol phosphate signal generation, whereas Met134 mutations also impaired receptor internalization. However, the D125A mutant AT1R exhibited moderate constitutive activity as indicated by increased basal ERK activation and enhanced inositol phosphate responses to partial agonists. Agonist-induced stimulation of the Elk1 promoter showed parallel impairment with inositol phosphate signal generation after mutations in this region of the AT1R. These data suggest that Ca2+ signaling is required for the nuclear effects of angiotensin II-induced ERK activation, and are consistent with the role of the conserved DRY sequence in AT1R activation, and of Asp125 in constraining the receptor in its inactive conformation. It is likely that an apolar surface that includes Ile130 and Met134 in the cytoplasmic extension of the third transmembrane helix has a direct role in G protein coupling and signal generation. We previously demonstrated that agonist-induced endocytosis of AT1 receptors is a clathrin- and dynamin-dependent process at physiological Ang II concentrations, but is independent of these proteins at high levels of agonist stimulation. Also, that Gq-mediated receptor signaling at the membrane level, and to MAP kinase cascades, is independent of receptor endocytosis. Once internalized, Ang II-receptor complexes are processed via endosomes to structures that resemble multivesicular bodies, and return to the cell surface by a rapid recycling pathway that is dependent on PI 3-kinase. The receptor is also recycled via a slower pathway that is less sensitive to inhibition of PI 3-kinase. In related studies, AT1 receptor endocytosis was also found to be dependent on the interaction of the proline-rich domain of dynamin-2 with SH3 domains of amphiphysins and endophilins, similar to the recruitment of dynamin-1 during the recycling of synaptic vesicles. In earlier studies we observed that agonist-induced internalization of the AT1 receptor is dependent on an STL triplet in its cytoplasmic C-terminal region, and is associated with phosphorylation of a specific serine/threonine-rich sequence in this domain. In yeast, hyperphosphorylation of the alpha-factor pheromone receptor has been reported to regulate endocytosis of the receptor by facilitating its monoubiquitylation at lysine residues in its cytoplasmic tail. To determine whether this process could contribute to the internalization of the AT1R, mutant receptors with deletion or replacement of lysine residues in their agonist-sensitive serine/threonine-rich region were expressed in CHO cells. These modifications had no effect on the Ang II-induced receptor endocytosis, and fusion of ubiquitin in-frame to an internalization-deficient truncated AT1 receptor mutant did not restore the endocytosis of the resulting chimeric receptor. No impairment of receptor internalization was observed after substitution of all lysine residues in the serine/threonine-rich region, performed at saturating angiotensin II concentrations where endocytosis occurs by a beta-arrestin- and dynamin-independent mechanism. These findings indicate that ubiquitylation of the AT1 receptor is not required for its agonist-induced internalization, and suggest that endocytosis of mammalian GPCRs occurs by different mechanisms than those of yeast GPCRs. However, it is possible that ubiquitylation has other functions in the biosynthesis and trafficking of the AT1R and other GPCRs.